In chemical analysis it is often useful to measure one or more of the optical properties of an unknown sample. Such techniques, particularly when used in combination with other techniques, can provide qualitative and quantitative information about an unknown sample. Before optical measurements are made, an analyst may first use a separation technique to separate a sample into its constituents. Well known separation techniques include gas chromatography (GC), high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). In systems employing these techniques a sample mixture is forced to flow through a separation column borne by a mobile phase. Different compounds in the sample move through the separation column at different speeds reaching the end of the column at different times. The elution of a sample component is commonly referred to as a "peak".
A variety of detection techniques and devices are available for qualitatively and quantitatively measuring a peak eluting from the end of a separation column. When using modern high resolution capillary separation techniques, a peak may consist of a very small mass of material which may elute over a relatively short time duration.
Various optical detection methods are in widespread use in HPLC, SFC and CE. Perhaps the most commonly used optical detection technique is absorbance detection wherein the optical absorbance of the sample flowing from the separation column is continuously measured. Fluorescence detection is another very commonly used technique. By measuring changes in the optical absorbance or fluorescence from background levels, qualitative and quantitative information about the sample may be obtained.
Generally, optical detection techniques used in connection with separation columns may be divided into two categories: those in which detection is performed "on-column" and those in which detection is performed using a flow-cell. On-column measurements are made by observing the properties of the sample near the end of the separation column but while the peak is still in the column. Flow-cells involve use of specially designed volumes for containing the flowing sample after it has eluted from the separation column. An example of a flow cell design for use in a liquid chromatography is shown in U.S. Pat. No. 4,006,990. Flow-cells can be designed to provide advantageous optical properties to enhance detection, but inherently add dead volume and cause fluid turbulence resulting in undesired peak broadening. On the other hand, on-column detection, while not suffering from the peak broadening problems associated with flow cells, does not offer very good optical properties.
The trend in separation science has been towards techniques capable of analyzing ever smaller samples. In the field of HPLC, this has resulted in the increased use of "microbore" or capillary columns. This trend has also led to increased use of SFC and CE, which are also performed using capillary columns. In systems using capillary columns sample volumes can be quite small. For example, to maintain high efficiency in CE, a sample plug must be less than 1 mm in length. Thus, when one uses a column having an inner diameter (ID) of 75 .mu.m, the detection volume is on the order of 4 nL. The small size of capillary columns and of the sample volumes associated with capillary columns exacerbates the problems associated with using flow cells. As a result, on-column detection has been the preferred method for use with capillary separation columns. A basic design for on-column detection in capillary column HPLC is shown in U.S. Pat. No. 4,375,163. This patent also contains a more thorough discussion of the limitations of flow cells when dealing with very small sample volumes.
On-column detection techniques have the disadvantage, described above, of not offering good optical properties. A major problem with on-column optical detection in connection with capillary columns is due to light which is scattered and thereafter interferes with detection. A beam of light incident on a column will interact not only with the fluid within the column but also with the surrounding glass wall. A typical capillary column may have, for example, an outer diameter of 375 .mu.m and an inner diameter of only 75 .mu.m. Thus, a cross section of such a column has a 75 .mu.m bore surrounded by two walls twice (150 .mu.m) as wide. If light is evenly incident on the entire column, the majority will interact with the column walls rather than the sample. Scattered light will not only cause non-linearity in detector response at high absorption, it will also cause a reduction from the true absorption at low absorption.
Accordingly, it is an object of the present invention to provide an improved on-column optical detection method and apparatus for use with capillary separation columns.
Another object of the present invention is to provide a method and apparatus which minimizes light scatter in an on-column optical detection system.
Yet another object of the present invention is to provide a method and apparatus which minimizes the adverse effects of scattered light in an on-column optical detection system employing a capillary column.
Still a further object of the present invention is to provide a unique lens design which achieves the foregoing objects.